Cell proliferation in CPC scaffold with a central channel

Calcium phosphate cement (CPC) scaffold design should improve nutrient and cell transfer to the scaffold centre. To achieve this goal, a channel network with proper channel diameters should be incorporated into the scaffold. In this study, CPC scaffolds with a single central channel were fabricated indirectly using a stereolithography rapid prototyping (RP) technology. The diameters of the central channels ranged from 402 microm to 1988 microm. These scaffolds were seeded with rabbit marrow stem cells (MSCs) labeling DiI and cultured for 5 days. Cell observation on the wall of the central channels was performed. The data of the experimental point revealed that cell coverage was from approximately 18% (1988 microm) to approximately 35% (592 microm). There was a significant increase from day 1 to day 5 in cell coverage in the same channel. The cell area coverage increased lineally with the central channel diameter when the channel diameter was less than approximately 789 microm. Afterwards (from 789 to 1988 microm), the relationship between cell area coverage and channel diameter was also linear relationship. But the increase was more slowly than before. Preliminary demonstration from the data that the minimum channel diameter required for cell migration into and adhesion on CPC scaffold was approximately 72 microm. These results are promising for the development of optimal scaffold with a three-dimensional channel network.     

In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel

There is a clinical need for synthetic scaffolds that promote bone regeneration. A common problem encountered when using scaffolds in tissue engineering is the rapid formation of tissue on the outer edge of the scaffold whilst the tissue in the centre becomes necrotic. To address this, the scaffold design should improve nutrient and cell transfer to the scaffold centre. In this study, hydroxyapatite scaffolds with random, open porosity (average pore size of 282+/-11microm, average interconnecting window size of 72+/-4microm) were manufactured using a modified slip-casting methodology with a single aligned channel inserted into the centre. By varying the aligned channel diameter, a series of scaffolds with channel diameters ranging from 170 to 421microm were produced. These scaffolds were seeded with human osteosarcoma (HOS TE85) cells and cultured for 8 days. Analysis of cell penetration into the aligned channels revealed that cell coverage increased with increasing channel diameter; from 22+/-3% in the 170microm diameter channel to 38+/-6% coverage in the 421microm channel. Cell penetration into the middle section of the 421microm diameter channel (average cell area coverage 121x10(3)+/-32x10(3)microm(2)) was significantly greater than that observed within the 170microm channel (average cell area coverage 26x10(3)+/-6x10(3)microm(2)). In addition, the data presented demonstrates that the minimum channel (or pore) diameter required for cell penetration into such scaffolds is approximately 80microm. These results will direct the development of scaffolds with aligned macroarchitecture for tissue engineering bone.     

Manufacture of porous polymer nerve conduits by a novel low-pressure injection molding process

A method to fabricate porous, biodegradable conduits using a combined injection molding, thermally induced phase transition technique was developed which produced conduits with dimensionally toleranced, longitudinally aligned channels. The geometry of the channels was designed to approximate the architecture of peripheral nerves and to support the monolayer adherence of physiologically relevant numbers of Schwann cells. The channel configuration could be varied from a single 1.35 mm diameter channel up to 100 0.08 mm diameter channels. A conduit with 100 channels has approximately 12.5 times the lumenal surface area of a single channel conduit and supports the adherence of five times the number of Schwann cells in the native peripheral nerve. In this study, poly(DL-lactide-co-glycolide) (DL-PLGA) was dissolved in acetic acid and injected into a cold mold which induced solid-liquid phase separation and, ultimately, solidification of the polymer solution. The acetic acid was removed by sublimation and the resulting foam had a macrostructure of high anisotropy. Semi-permeable skins formed on the outer and lumen diameters of the conduit as a consequence of rapid quenching. Macropores were organized into bundles of channels, up to 20 microm wide, in the DL-PLGA matrix and represented remnants of acetic acid that crystallized during solidification.     

Resorbable polymeric scaffolds for bone tissue engineering: the influence of their microstructure on the growth of human osteoblast-like MG 63 cells

Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.     

Tailoring fiber diameter in electrospun poly(epsilon-caprolactone) scaffolds for optimal cellular infiltration in cardiovascular tissue engineering

Despite the attractive features of nanofibrous scaffolds for cell attachment in tissue-engineering (TE) applications, impeded cell ingrowth has been reported in electrospun scaffolds. Previous findings have shown that the scaffold can function as a sieve, keeping cells on the scaffold surface, and that cell migration into the scaffold does not occur in time. Because fiber diameter is directly related to the pore size of an electrospun scaffold, the objective of this study was to systematically evaluate how cell delivery can be optimized by tailoring the fiber diameter of electrospun poly(epsilon-caprolactone) (PCL) scaffolds. Five groups of electrospun PCL scaffolds with increasing average fiber diameters (3.4-12.1 microm) were seeded with human venous myofibroblasts. Cell distribution was analyzed after 3 days of culture. Cell penetration increased proportionally with increasing fiber diameter. Unobstructed delivery of cells was observed exclusively in the scaffold with the largest fiber diameter (12.1 microm). This scaffold was subsequently evaluated in a 4-week TE experiment and compared with a poly(glycolic acid)-poly(4-hydroxybutyrate) scaffold, a standard scaffold used successfully in cardiovascular tissue engineering applications. The PCL constructs showed homogeneous tissue formation and sufficient matrix deposition. In conclusion, fiber diameter is a crucial parameter to allow for homogeneous cell delivery in electrospun scaffolds. The optimal electrospun scaffold geometry, however, is not generic and should be adjusted to cell size.     

Three-dimensional ingrowth of bone cells within biodegradable cryogel scaffolds in bioreactors at different regimes

Three-dimensional cell ingrowth within biodegradable cryogel scaffolds made of cross-linked 2-hydroxyethyl methacrylate (HEMA)-lactate-dextran with interconnected macropores was studied in bioreactors at different regimes (static, perfusion, and compression-perfusion). An osteoblast-like cell line (MG63) was used in these studies. The samples taken after selected times from the bioreactors were examined by microscopy techniques (light, SEM, TEM, and laser scanning confocal). The cell culture conditions were found to have a significant impact not only on the cell morphology, such as the extent of cell attachment and ingrowth, but also on cellular activities. Dynamic conditions (perfusion and/or compression) greatly improved cell ingrowth and extracellular matrix (ECM) synthesis. Alkaline phosphatase activity results confirmed the positive effect of dynamic conditions on bone cells.     

Optimising bioactive glass scaffolds for bone tissue engineering

A 3D scaffold has been developed that has the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. Sol-gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The scaffolds consist of a hierarchical pore network with macropores in excess of 500 microm connected by pore windows with diameters in excess of 100 microm, which is thought to be the minimum pore diameter required for tissue ingrowth and vasularisation in the human body. The scaffolds also have textural porosity in the mesopore range (10-20 nm). The scaffolds were sintered at 600, 700, 800 and 1000 degrees C. As sintering temperature was increased to 800 degrees C the compressive strength increased from 0.34 to 2.26 MPa due to a thickening of the pore walls and a reduction in the textural porosity. The compressive strength is in the range of that of trabecular bone (2-12 MPa). Importantly, the modal interconnected pore diameter (98 microm) was still suitable for tissue engineering applications and bioactivity is maintained. Bioactive glass foam scaffolds sintered at 800 degrees C for 2 h fulfill the criteria for an ideal scaffold for tissue engineering applications.     

Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration

The human amnion consists of the epithelial cell layer and underlying connective tissue. After removing the epithelial cells, the resulting acellular connective tissue matrix was manufactured into thin dry sheets called amnion matrix sheets. The sheets were further processed into tubes, amnion matrix tubes (AMTs), of varying diameters, with the walls of varying numbers of amnion matrix sheets with or without a gelatin coating. The AMTs were implanted into rat sciatic nerves. Regenerating nerves extended in bundles through tubes of 1-2 mm in diameter and further elongated into host distal nerves 1-3 weeks after implantation. Morphometrical analysis of the regenerated nerve cable at the middle of each amnion matrix tube 3 weeks after implantation was performed. The average numbers of myelinated axons were almost the same (ca. 80-112/10(4) microm(2)) in AMTs of 1-2 mm in diameter, as in the normal sciatic nerve (ca. 95/10(4) microm(2)). No myelinated fibers were found in AMTs composed of multiple thin tubes of 0.2 mm in diameter. The myelinated axons were thinner in implanted tubes than those in the normal sciatic nerve. The rate of occurrences of myelinated axons less than 4 microm in diameter was significantly higher in the AMTs, whereas axons in the normal sciatic nerve were diverse in distribution, with the highest population at 8-12 microm in diameter. Reinnervation to the gastrocnemius muscle was demonstrated electrophysiologically 9 months after implantation. It was concluded that the extracellular matrix sheet from the human amnion is an effective conduit material for peripheral nerve regeneration.     

Effects of different titanium alloys and nanosize surface patterning on adhesion, differentiation, and orientation of osteoblast-like cells

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced patterns of parallel Ti oxide lines with different widths (0.2-10 microm) and distances (2-20 and 1,000 microm), but a common height of only 12 nm. These structures did not influence ALP activity (days 5-9), but had a positive effect on cell alignment. Two days after plating, the majority of the focal contacts were placed on the oxide lines. The portion of larger focal adhesions bridging two lines was inversely related to the line distance (2-20 microm). In contrast, the portion of aligned cells did not depend on the line distance. On average, 43% of the cells orientated parallel towards the lines, whereas 34% orientated vertically. In the control pattern (1,000 microm line distance), cell distribution was completely at random. Because a significant surplus of the cells preferred a parallel alignment, the nanosize difference in height between Ti surface and oxide lines may be sufficient to orientate the cells by contact guiding. However, gradients in electrostatic potential and surface charge density at the Ti/Ti oxide interface may additionally influence focal contact formation and cell guidance.     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

The attachment and growth behavior of osteoblast-like cells on microtextured surfaces

In previous studies, we showed that the application of microgrooves on a surface can direct cellular morphology and the deposition of mineralized matrix of osteoblast-like cells (Biomaterials 20 (1999) 1293; Clin. Oral Impl Res. 11 (2000) 325). In this study, we evaluated the attachment and growth behavior of these cells, using scanning- and transmission electron microscopy (SEM/TEM). Smooth and microgrooved polystyrene substrates were made (groove depth 0.5-1.5 microm, groove- and ridge width 1-10 microm). On these substrates, osteoblast-like cells were cultured for periods up to 16 days. SEM showed that the cells, and their extensions, closely followed the surface on smooth and wider grooved (>5 microm) substrates. In contrast, narrow grooves (<2 microm) were bridged. After 16 days of incubation, the matrix showed extensive deposition of collagen fibrils, and the formation of calcified nodules. With TEM it was shown that on the smooth and wider grooved substrates, focal adhesions were spread throughout the surface. However, on narrow grooves focal adhesions were always positioned on the edges of surface ridges only. Apparently, most extracellular matrix (ECM) was produced by the cells that directly adhered to the substrate. Deposition of ECM was seen in the surface grooves, as well as in between the cell layers. On basis of the current study and previous experiments, we conclude that microgrooves are able to influence bone cell behavior by (1) determining the alignment of cells and cellular extensions, (2) altering the formation and placement of cell focal adhesions, and (3) altering ECM production. Therefore, microgrooved surfaces seem interesting to be applied on bone-anchored implants.     

Silicon-substituted hydroxyapatite thin films: effect of annealing temperature on coating stability and bioactivity

The effect of annealing temperature on the physicochemical and biological characteristics of magnetron cosputtered silicon-substituted hydroxyapatite (SiHA) thin coatings was studied. Annealing is required to transform as-sputtered amorphous films into crystalline coatings. A nanocrystalline, single-phase apatite structure was achieved for coatings heated to 600 or 700 degrees C and, with increasing annealing temperature, the crystallite size increased. Small crystallites were found to be more soluble in the physiological environment but, at the same time, were able to induce early formation of a new apatite layer. A human osteoblast-like (HOB) cell model was used to evaluate the performance of these annealed SiHA coatings. HOB cells attached and grew well on coatings and, after 42 days in culture, a mineralization process was observed to be taking place, with evidence of calcium phosphate minerals throughout the extracellular matrix. Our findings indicated that an annealing temperature of 600 degrees C is sufficient to achieve crystalline SiHA coatings and exhibiting good chemical stability and bioactivity.     

Effect of high hydrostatic pressure on biological properties of extracellular bone matrix proteins

In orthopedic surgery, sterilization of bone used for reconstruction of osteoarticular defects caused by malignant tumors is carried out in various ways. At present, to devitalize tumor-bearing osteochondral segments, extracorporal irradiation or autoclaving is mainly used but both methods have substantial disadvantages, for instance, loss of biomechanical and biological integrity of the bone. In particular, after reimplantation, integration of the implant at the autograft-host junction is often impaired due to alteration of osteoinductivity as a result of its irradiation or autoclaving. As an alternative approach, high hydrostatic pressure (HHP) treatment of bone is suggested, a new technology which is in the preclinical testing stage, with the aim to inactivate tumor cells but leaving the biomechanical properties of bone, cartilage, and tendons intact. We investigated the influence of HHP on the major extracellular matrix (ECM) proteins, fibronectin (FN), vitronectin (VN), and type I collagen (Col-I), present in bone tissue, which are accountable for the biological properties within the bone. FN, VN, and Col-I were subjected to HHP < or = 600 MPa prior to coating of cell culture plates with these matrix proteins. Thereafter, the capacity of HHP-pretreated FN, VN, and Col-I to affect cell proliferation, cell adherence, and spreading of human primary osteoblast-like cells and the human osteosarcoma cell line Saos-2, was tested. Interestingly, even at HHP < or = 600 MPa, all three ECM proteins retained their biological properties because no significant changes were observed between HHP-treated and non-treated FN, VN, and Col-I regarding their biological properties to affect cell adherence, spreading, and proliferation. These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to reimplantation. While during this treatment eukaryotic cells including tumor cells will be irreversibly impaired, the bone's biomechanical properties and the biological properties of the ECM proteins FN, VN, and Col-I, respectively, are preserved.     

Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants

Three-dimensional macroporous calcium phosphate bioceramics embedded with porous chitosan sponges were synthesized to produce composite scaffolds with high mechanical strength and a large surface/volume ratio for load-bearing bone repairing and substitutes. The macroporous calcium phosphate bioceramics with pore diameters of 300 microm to 600 microm were developed using a porogen burnout technique, and the chitosan sponges were formed inside the pores of the bioceramics by first introducing chiosan solution into the pores followed by a freeze-drying process. Our scanning electron microscopy results showed that the pore size of chitosan sponges formed inside the macroporous structure of bioceramics was approximately 100 microm, a structure favorable for bone tissue in-growth. The compressive modulus and yield stress of the composite scaffolds were both greatly improved in comparison with that of HA/beta-TCP scaffolds. The simulated body fluid (SBF) and cell culture experiments were conducted to assess the bioactivity and biocompatibility of the scaffolds. In the SBF tests, a layer of randomly oriented needle-like apatite crystals formed on the scaffold surface after sample immersion in SBF, which suggested that the composite material has good bioactivity. The cell culture experiments showed that MG63 osteoblast cells attached to the composite scaffolds, proliferated on the scaffold surface, and migrated onto the pore walls, indicating good cell biocompatibility of the scaffold. The cell differentiation on the composite scaffolds was evaluated by alkaline phosphatase (ALP) assay. Compared with the control in tissue culture dishes, the cells had almost the same ALP activity on the composite scaffolds during the first 11 days of culture.     

Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells

Objectives: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. Materials and methods: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. Results: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. Conclusions: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion.     

Microfluidic channel‐assisted screening of hematopoietic malignancies

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro‐channel, which resembled a 250‐fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50‐fold less probe consumption and 10‐fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro‐channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over‐score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH. © 2013 Wiley Periodicals, Inc.     

The structural and biological properties of hydroxyapatite-modified titanate nanowire scaffolds

Hydroxyapatite-modified titanate nanowire scaffolds as alternative materials for tissue engineering have been developed via a titanate nanowire matrix assisted electrochemical deposition method. The macroporous titanate nanowire matrix on Ti metal was fabricated by a hydrothermal method, and then followed by an electrochemical synthesis of hydroxyapatite nanoparticles on titanate nanowire. The incorporation of titanate nanowire matrix with high oriented hydroxyapatite nanoparticles generates hierarchical scaffolds with highly osteogenic, structural integrity and excellent mechanical performance. As-prepared porous three dimensional interconnected hydroxyapatite-modified titanate nanowire scaffolds, mimicking the nature's extracellular matrix, could provide a suitable microenvironment for tissue cell ingrowth and differentiation. The ceramic titanate nanowire core with HA nanoparticle sheath structure displays superhydrophilicity, which facilitates the cell attachment and proliferation, and induces the in vitro tissue-engineered bone. Human osteoblast-like MG63 cells were cultured on the hydroxyapatite-modified titanate nanowire scaffolds, and the results showed that the scaffolds highly promote the bioactivity, osteoconductivity and osteoblast differentiation.     

Mechanical stimuli induce intracellular calcium response in a subpopulation of cultured rat sensory neurons.

Cultured dorsal root ganglion neurons from newborn rats were mechanically deformed with a fine-tipped glass capillary, and the change in the intracellular Ca2+ concentration ([Ca2+]i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca2+]i from 18.7 +/- 5.4 nM (mean +/- S.E.M., n = 35) to 137.1 +/- 15.2 nM in some subpopulations of cells, especially those larger than 20 microm in diameter. The largest mechanosensitive cell group was that of cells 20-25 microm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 microm in diameter displayed the Ca2+ increase when prodded. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca2+ channels, did not influence the Ca2+ response, whereas gadolinium, a relatively selective blocker of mechanosensitive channels, diminished the response. Na+-free conditions did not influence the response. We concluded that the mechanical stimulation induced a Ca2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca2+-permeable channels.     

Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide)

The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.